Fluorescently labeled probes provide a convenient method of characterizing the content of biological samples. By tailoring the binding chemistry of a fluorescent probe, high specificity can be achieved for detection of complex molecules such as RNA, DNA, proteins, and cellular structures. Since fluorophores typically absorb and re-emit Stokes-shifted radiation regardless of being bound or unbound to a species to be detected, the bound and unbound fluorophores must be separated.
One common method to separate the bound fluorophores from the unbound fluorophores relies on spatial localization of the fluorescently labeled species. For example, in a ‘sandwich immunoassay,’ a surface is chemically treated to bind a species to be detected to that surface. The fluorescent probes then attach to the species that are bound to the surface. Unbound fluorophores can then be removed from the system with a wash step.
Background fluorescence can be further reduced if the excitation light can be confined to the surface. Total internal reflection fluorescence (TIRF) is one method of reducing background fluorescence. In general, when light propagates from one medium to another, a portion of the light will be reflected at the interface. If the light is propagating into a material with a lower index of optical refraction, however, all of the light will be reflected if the angle at which the beam is incident on the surface is greater than the ‘critical angle’ (relative to the surface normal). In the lower index material, the light intensity exponentially decays with distance from the surface. This exponentially decaying field (known as an ‘evanescent field’) has a characteristic decay length on the order of 100 nanometers to 1 micrometer for visible light. The light of the evanescent field will, therefore, only excite fluorophores that are localized at the surface.
In a simplified implementation, TIRF is performed with a laser beam reflecting once from the surface. This is the basis of well established TIRF microscopy and other biosensing techniques. By confining the laser beam inside a waveguide, however, multiple reflections can be realized and larger areas can be illuminated. Several waveguide geometries are possible, each having certain tradeoffs.
Single-mode planar waveguides, also called thin film waveguides or integrated optical waveguides, confine light into a small cross sectional area with the thin dimension smaller than the wavelength of propagating light. The advantage of single-mode waveguides is that significantly stronger evanescent fields are generated. A disadvantage of single-mode waveguides is that for efficient light coupling, they typically require a prism or grating with precise alignment tolerances. In addition, single-mode planar waveguides are expensive to manufacture because the guiding layer is typically a thin-film with strict thickness tolerances deposited on a substrate. In contrast, a multimode planar waveguide is substantially easier to couple a laser beam to and simpler to construct than single-mode planar waveguides. For example, a standard 1 millimeter thick microscope slide makes an effective waveguide into which light can be coupled through the edge of the slide. Additionally, dimensions for multimode waveguides are compatible with current plastic injection-molding techniques.
For a fluorescence-based assay system, a uniform evanescent field is desired in the detection region. By definition, the strength of the evanescent field is uniform along the direction of light propagation for a single-mode planar waveguide (neglecting scattering losses and absorption inside the waveguide). For a disposable clinical device, however, cost, robustness, and ease of use are of similar importance. By adjusting input coupling to a multimode waveguide, uniformity and field strength of the evanescent field can be optimized.
While each individual mode in a multimode waveguide has a uniform intensity along the direction of propagation, a distribution of modes will be excited when coupling to a multimode waveguide; this distribution of modes will constructively and destructively interfere on the surface and lead to a spatially varying field strength. When the thickness of the waveguide is much larger than the wavelength of light, the mode structure of the waveguide can be neglected, and the intensity in the waveguide can be treated as a conventional diffracting beam that totally-internally reflects from the two surfaces of the waveguide and interferes with neighboring reflections.
FIG. 1 illustrates several examples of existing coupling schemes 105-115 involving multimode waveguides. Coupling scheme 105 using a multimode waveguide 120 involves focusing a laser beam 125 that propagates parallel to a waveguide 120 into the edge of waveguide 120 with a cylindrical lens 130. The field strength of a total internal reflection (“TIR”) beam, however, is maximized for a beam that is incident at the critical angle and zero for a beam with an incident angle 90° from the surface normal (i.e., grazing incidence). Thus, an incident beam that is parallel to the TIR surface will have small evanescent field strength when coupled to waveguide 120 with cylindrical lens 130 in the configuration of the scheme 105.
A variation on coupling scheme 105 is illustrated by coupling scheme 110. In coupling scheme 110, a laser beam 135 focused by a cylindrical lens 140 is incident on the edge of a waveguide 145 with an appropriate angle such that a central ray of laser beam 135 inside the waveguide impinges on the surface near the critical angle for TIR to maximize the evanescent field strength. A compromise between field strength and uniformity may be made by the choice of focusing optics. If a nearly collimated beam is used to achieve high field intensity by operating near the critical angle for TIR, the beam must make many reflections within the waveguide before the surface intensity becomes sufficiently uniform, thus requiring a longer waveguide. If the beam is highly focused, however, then the surface intensity normalizes in very few reflections, but a significant amount of power is contained in rays propagating outside the critical angle and leads to reduced evanescent field strength down the length of the waveguide.
Precise alignment of a cylindrical lens, such as lenses 130 and 140, relative to the input face of a waveguide, such as waveguides 120 and 145, respectively, must be made in order to have a laser beam focused on the input face. One proposed solution to this problem is illustrated by a coupling scheme 115. In coupling scheme 115, a lens 150 is incorporated with a waveguide 155 as a single optical component, made, for example, by bonding the lens element to the planar waveguide or by molding a single optical component. While this allows the focus of lens 150 to be precisely distanced from the edge of waveguide 155, careful alignment of a laser beam 160 relative to lens 150 of waveguide 155 must still be made to couple beam 160 to waveguide 155. For applications requiring repeated placement of a waveguide component relative to the light source, it is highly desirable for the light coupling to be relatively insensitive to misalignment.
In practical applications, the penetration depth of the evanescent field usually is less than a wavelength of the incident light. This aspect is an advantage in some applications, as the evanescent field can serve as a mechanism to illuminate only a volume of interest, e.g., a thin layer in the lower refractive index medium proximate to the waveguide surface. On the other hand, when the object of interest, such as a cell or the bulk of a solution, extends substantially beyond the penetration depth of the evanescent wave, evanescent illumination can be less effective than floodlight-type illumination.
A subfield of integrated optofluidics is concerned with the development of methods for using optical waveguides to illuminate extended liquid media. Most of the developed methods involve the containment of a liquid sample by other liquid and/or solid materials, thereby effectively creating a waveguide for illuminating the liquid sample. Most TIR-based designs involve surrounding the liquid sample with media of lower index of refraction than that of the liquid sample itself. It is then theoretically possible for light to be guided in the liquid sample by TIR at the interface between the high refractive index liquid and the lower refractive index surroundings. However, in practice, waveguiding in a liquid sample contained in another material is difficult due to the fact that common liquids have lower refractive indices than common solids; for example, water has a refractive index of approximately 1.33, while most solid materials have an index of refraction of 1.4 or more. Consequently, a majority of the TIR waveguide designs involve using either high refractive index (i.e., “high-n”) liquids or more exotic low refractive index (i.e., “low-n”) solids.
In interference-based optofluidic waveguides, light is confined to a liquid core by reflection from surrounding materials including two or more layers of higher-index materials combined to result in a lower effective refractive index for the surrounding media. Some interference-based optofluidic waveguides include photonic crystals, such as multiple alternating layers of materials of different indices of refraction